Human cytomegalovirus (“HCMV”) infects between 50% and 85% of adults by 40 years of age (Gerson A. A., et al., in Viral Infections of Humans, Evans A. S. and Kaslow, R. A., eds., Plenum Press, New York, N.Y. (1997)). Although HCMV infection is benign in most healthy adults, it can result in deadly pneumonitis, as well as colitis, esophagitis, leukopenia, and retinitis in transplant and other immunocompromised patients, especially those with HIV. In solid organ transplant (SOT) or hematopoeitic cell transplant (HCT) populations, HCMV disease can occur either from new infection transmitted from the donor organ or HCT, or can recur as a result of reactivation of latent virus in the recipient.
Despite licensed therapies, HCMV-associated disease remains severely debilitating and life-threatening in HIV patients and the allogeneic related HCT and SOT settings. In addition, HCMV is the most common intrauterine infection in the United States, and results in death or severe sequelae in over 8,000 infants per year. For these reasons, HCMV was ranked in the list of the top 10 vaccines most in need of development in the United States (Vaccines for the 21st century: a tool for decision making, National Academy of Sciences (1999)).
Existing therapies include the use of immunoglobulins and anti-viral agents such as ganciclovir and its derivatives, which are most effective when used prophylactically or very early during infection in at risk populations. However, these therapies are characterized by significant toxicity and limited efficacy, especially for late onset disease (onset after the first 100 days) (Fillet, A. M., Drugs Aging 19:343-354 (2002); von Bueltzingsloewen, A., et al., Bone Marrow Transplant 12:197-202 (1993); Winston, D. J., et al., Ann. Intern. Med. 118:179-184 (1993); Goodrich, J. M., et al., Ann. Intern. Med. 118:173-178 (1993); Boeckh, M., et al., Blood 88:4063-4071 (1996); Salzberger, B., et al., Blood 90:2502-2508 (1997); Preiser, W., et al., J. Clin. Virol. 20:59-70 (2001); Grangeot-Keros, L., and Cointe, D., J. Clin. Virol. 21:213-221 (2001); Boeckh, M., and Bowden, R., Cancer Treat. Res. 76:97-136 (1995); Zaia, J. A., et al., Hematology (Am. Soc. Hematol. Educ. Program) 339-355 (2000)).
In addition to developing more rapid and sensitive diagnostics, molecular biological methods enable the development of defined subunit vaccines for human pathogens. Indeed, safe, effective recombinant subunit vaccines would significantly reduce, and perhaps eliminate, the need for therapeutic treatments. In the case of HCMV, control of infection has been correlated with antibody and T cell recognition of at least three viral proteins: pp65, glycoprotein B (gB), and the immediate early-1 protein (IE1).
The 65kD viral protein pp65, also known as ppUL83, lower matrix protein, ICP27, PK68, and pp64, is one of the most abundantly expressed structural proteins (FIG. 1). It is encoded by the UL83 gene of the viral genome (nucleotides 119352-121037 of the HCMV strain AD169 genomic sequence, Genbank X17403). This protein is believed to be processed for MHC presentation shortly after viral entry into cells, which allows it to be presented before other viral proteins shut down the antigen processing pathway in infected cells. Therefore, T cell recognition of this protein is important for infection control (Solache, et al. J. Immunol. 163:5512-5518 (1999)), which is herein incorporated by reference in its entirety.
Glycoprotein B (gB) is a 906 amino acid envelope glycoprotein (FIG. 4) encoded by UL55, nucleotides 80772-83495 of Genbank X17403). It is a type I integral membrane protein that participates in the fusion of the virion envelope with the cell membrane, is required for infectivity, is highly immunogenic, and has a high degree of conservation among HCMV strains, making this protein an attractive target for vaccines. The full-length protein contains an amino-terminal signal peptide (amino acids 1-24), an extracellular domain (amino acids 25-713), a putative trans-membrane anchor domain (amino acids 714-771) and an intracellular domain (amino acids 772-906). Deletion of the transmembrane anchor domain results in secretion of gB (Zheng et al. J. Virol. 70:8029-8040 (1996)). Additionally, the full-length protein is cleaved by host furin proteases between amino acids 460 and 461 to form the gp93 and gp55 cleavage products that remain tightly associated as a heterodimer. (Mocarski E. S. and C. T. Courcelle, pp. 2629-2674, Field's Virology, 4th ed., Eds. Knipe D M and Howley P M, Lippincott Williams & Wilkins, Philadelphia (2001)). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
IE1 is a 491 amino acid protein (FIG. 7) encoded by HCMV ORF UL123 (Genbank X17403, nucleotides 171006-172765). The gene encodes a 1.9 Kb mRNA comprising four exons, with only exons 2-4 being translated. The 85 N-terminal amino acids are encoded by exons 2 and 3, with the remainder encoded by exon 4. IE2 is a related family of proteins that share exons 1-3 and an exon 5, with many splice variations. Together, IE1 and IE2 transactivate the HCMV major immediate early promoter to regulate viral transcription (Malone, C L. et al. J. Virol. 64:1498-1506 (1990); Mocarski, E. Fields Virology Ed. Field et al., 3rd ed., pp. 2447-2491, Lippincott-Raven Publishers, Philadelphia (1996); Chee M. S. et al., Curr Topics Microbiol. Immunol. 154:125-169 (1990)). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
IE1 has a kinase activity that is dependent on an ATP binding site encoded by amino acids 173-196. IE1 can autophosphorylate or phosphorylate cellular factors to transactivate E2F dependent transcription. Both exons 3 and 4 are required for viral transactivation, with the required regions in exon 4 being broadly distributed throughout the exon. The portion of the protein encoded by exon 4 is known to have a high degree of secondary structure. Although IE1 is transported to the nucleus, no nuclear localization signal has been identified. (Pajovic, S. et al. Mol. Cell. Bio. 17:6459-6464 (1997)). Gyulai et al. showed high levels of CTL response in vitro to effector cells expressing a nucleotide fragment consisting of exon 4 (Gyulai et al. J. Infectious Diseases 181:1537-1546 (2000)). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
No vaccine is currently available for HCMV. However, clinical trials have been performed with live-attenuated HCMV vaccines, a canarypox-based vaccine, and a recombinant gB vaccine (Plotkin, S. A., Pediatr. Infect. Dis. J. 18:313-325 (1999)). The first HCMV vaccine tested in humans was a live attenuated virus vaccine made from the AD169 laboratory-adapted strain (Elek, S. D. and Stern, H., Lancet 1:1-5 (1974)). Local reactions were common, but HCMV was not isolated from any of the vaccine recipients. This vaccine was not investigated beyond initial Phase I studies.
Immune responses to HCMV have been determined by the study of acute and chronic HCMV infections in both animal models and in man. Antibody appears critical in the prevention of maternal-fetal transmission, and is primarily directed to the envelope glycoproteins, especially gB (Plotkin, S. A., Pediatr. Infect. Dis. J. 18:313-325 (1999); Fowler, K. B., N. Engl. J. Med. 326:663-667 (1992)).
In contrast, the control of HCMV infection in transplant recipients and HIV-infected persons is associated with preserved cellular immune responses, including CD4+, CD8+, and NK T cells. The CD8+ T-cell responses are directed primarily at the immediate early (IE) protein of HCMV and at the abundant tegument protein pp65 (Gyulai, Z., et al., J. Infect. Dis. 181:1537-1546 (2000); Tabi, Z., et al., J. Immunol. 166:5695-5703 (2001); Wills, M. R., et al., J. Virol. 70:7569-7579 (1996); Frankenberg, N., et al., Virology 295:208-216 (2002); Retiere, C., et al., J. Virol. 74:3948-3952 (2000); Koszinowski, U. H., et al., J. Virol. 61:2054-2058 (1987); Kern, F., et al., J. Infect. Dis. 185:1709-1716 (2002)). Approximately 92% of persons have CD8+ responses to pp65 and another 76% to exon 4 of IE1 (Gyulai, Z., et al., J. Infect. Dis. 181:1537-1546 (2000); Kern, F., et al., J. Infect. Dis. 185:1709-1716 (2002)). In addition, another one third of infected individuals have CTL responses to gB. Almost all infected persons have CD4+ responses to HCMV, although the gene and epitope mapping of these responses is not as fully investigated as those for CD8+ T cells (Kern, F., et al., J. Infect. Dis. 185:1709-1716 (2002); Davignon, J. L., et al., J. Virol. 70:2162-2169 (1996); He, H., et al., J. Gen. Virol. 76:1603-1610 (1995); Beninga, J., et al., J. Gen. Virol. 76:153-160 (1995). The helper T-cell responses in infected, healthy persons are sufficiently robust that HCMV is frequently used as a positive control in the development of methods for the measurement of CD4+ T-cell responses (Kern, F., et al., J. Infect. Dis. 185:1709-1716 (2002); Currier, J. R., et al., J. Immunol. Methods 260:157-172 (2002); Picker, L. J., et al., Blood 86:1408-1419 (1995)).
Other attempts to develop vaccines for HCMV have focused on administering purified or recombinant viral polypeptides, either full-length, modified, or short epitopes, to induce immune responses. In a review published by the American Society for Hematology, Zaia et al. describes various peptide-based approaches to developing HCMV vaccines, including using DNA vaccines to express wild-type and mutated proteins (Zaia, J. A. et al. Hematology 2000, Am Soc Hematol Educ Program, pp. 339-355, Am. Soc. Hematol. (2000)). Endresz et al. describes eliciting HCMV-specific CTL in mice immunized with plasmids encoding HCMV Towne strain full-length gB, expressed constitutively or under a tetracycline-regulatable promoter, and pp65 or a gB with the deletion of amino acids 715-772 (Endresv, V. et al. Vaccine 17:50-8 (1999); Endresz, V. et al. Vaccine 19:3972-80 (2001)). U.S. Pat. No. 6,100,064 describes a method of producing secreted gB polypeptides lacking the transmembrane domain but retaining the C terminal domain. U.S. Pat. Nos. 5,547,834 and 5,834,307 describe a gB polypeptide with amino acid substitutions at the endoproteolytic cleavage site to prevent proteolytic processing. U.S. Pat. Nos. 6,251,399 and 6,156,317 describe vaccines using short peptide fragments of pp65 comprising immunogenic epitopes. A number of other groups have analyzed epitopes in HCMV pp65 and gB for eliciting a strong immune response (Liu, Y N. et al. J. Gen. Virol. 74:2207-14 (1993); Ohlin, M. et al. J. Virol. 67:703-10 (1993); Navarro, D. et al. J. Med. Virol. 52:451-9 (1997); Khattab B A. et al. J. Med. Virol. 52:68-76 (1997); Diamond, D J. et al. Blood 90:1751067 (1997); Solache, A. et al. J. Immunol. 163:5512-8 (1999). U.S. Pat. No. 6,162,620 is directed to a polynucleotide encoding a wild-type gB or a gB lacking the membrane sequences. U.S. Pat. No. 6,133,433 is directed to a nucleotide encoding a full-length, wild-type pp65 or a specific 721 nt fragment thereof. Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
During the past few years there has been substantial interest in testing DNA-based vaccines for a number of infectious diseases where the need for a vaccine, or an improved vaccine, exists. Several well-recognized advantages of DNA-based vaccines include the speed, ease and cost of manufacture, the versatility of developing and testing multivalent vaccines, the finding that DNA vaccines can produce a robust cellular response in a wide variety of animal models as well as in man, and the proven safety of using plasmid DNA as a delivery vector (Donnelly, J. J., et al., Annu. Rev. Immunol. 15:617-648 (1997); Manickan, E., et al., Crit. Rev. Immunol. 17(2):139-154 (1997)). DNA vaccines represent the next generation in the development of vaccines (Nossal, G., Nat. Med. 4:475-476 (1998)) and numerous DNA vaccines are in clinical trials. Each of the references cited in this paragraph is incorporated herein by reference in its entirety.
The immunotherapeutic product design is based on the concept of immunization by direct gene transfer. Plasmid-based immunotherapeutics offer the positive attributes of immune stimulation inherent to live-attenuated vaccines combined with the safety of recombinant subunit vaccines in an adjuvant formulation.
In the transplant population, control of HCMV disease is associated with a cellular immune response (Riddell, S. R., “Pathogenesis of cytomegalovirus pneumonia in immunocompromised hosts,” Semin. Respir. Infect. 10:199-208 (1995)) and thus an effective product should induce CD4+ and CD8+ T-cell responses. Formulated plasmid has been shown to induce such cellular immune responses, and does not have the safety concerns associated with the use of live vectors in the transplant setting (Shiver, J. W., et al., Nature 415:331-335 (2002)).
Retooling coding regions encoding polypeptides from pathogens using codon frequencies preferred in a given mammalian species often results in a significant increase in expression in the cells of that mammalian species, and concomitant increase in immunogenicity. See, e.g., Deml, L., et al., J. Virol. 75:10991-11001 (2001), and Narum, D L, et al., Infect. Immun. 69:7250-7253 (2001), all of which are herein incorporated by reference in its entirety.
There remains a need in the art for convenient, safe, and efficacious immunogenic compounds to protect humans against HCMV infection. The present invention provides safe yet effective immunogenic compounds and methods to protect humans, especially transplant recipients and immunocompromised individuals, against HCMV infection using such immunogenic compounds.